The objective of this study was to determine the optimal conditions under which to assess glucose tolerance in chow and high-fat fed C57BL/6J mice. Mice were fed either chow or high fat diet for 8 weeks. Variables tested were: fasting duration (0, 3, 6, overnight and 24 hrs fasting), route of administration (intraperitoneal vs oral) load of glucose given (2 g/kg, 1 g/kg or 0.5 g/kg and fixed 50 mg dose) and state of consciousness. Basal glucose concentrations were increased in high-fat compared to chow fed mice following 6 hrs of fasting (9.1 ± 0.3 vs. 7.9 ± 0.4 mmol/l p=0.01). Glucose tolerance was most different and therefore significant (p=0.001) in high-fat fed mice after 6 hrs of fasting (1973 ± 96 vs. 1248 ± 83 mmol/l x 120min). The difference in glucose tolerance was greater following an OGTT (142%), in contrast to an IPGTT with a 127% difference between high-fat and chow. We also found that administering 2 g/kg of glucose resulted in a greater level of significance (p=0.0008) in glucose intolerance in high-fat compared to chow fed mice. A fixed dose of 50 mg glucose regardless of body weight was enough to show glucose intolerance in high-fat vs chow fed mice. Finally, high-fat fed mice showed glucose intolerance compared to their chow counterparts whether tested under conscious or anesthetized conditions. We conclude that a 2 g/kg of glucose administered orally following 6 hrs of fasting is best to assess glucose tolerance in mice under these conditions.